PROJECT SUMMARY/ABSTRACT MOLECULAR REGULATION OF CARDIOVASCULAR SEVEN TRANSMEMBRANE RECEPTORS. All aspects of cardiovascular function are regulated by receptors of the seven transmembrane receptor (7TMR or GPCR) family, and they are the commonest target of therapeutic drugs. A universal mechanism regulating these receptors is desensitization of heterotrimeric G protein signaling. Classically, this is mediated by a two- step process in which activated receptors are phosphorylated by G protein-coupled receptor kinases, leading to the binding of a ?-arrestin (?arr) molecule which sterically interdicts further activation of the G protein. More recently it has become clear that ?arrs can also serve as multifunctional adaptors which act as signal transducers in their own right. Moreover, for many receptors ligands can be found which disproportionately activate either G protein- or ?arr-mediated signaling?i.e., biased ligands which may possess greater specificity of action and fewer side effects. Several such drugs, including one for decompensated congestive heart failure which targets the angiotensin II type 1 receptor (AT1R), have reached clinical trials. Accordingly, this proposal has three closely linked aims which involve developing a molecular- and atomic-level understanding of how such ?arr-mediated signaling is generated using as model systems two receptors of great cardiovascular significance, the ?2-adrenergic receptor (?2AR) and the AT1R. These aims are: 1) To discover novel allosteric stabilizers (nanobodies and small molecules) for biased conformations of the AT1R and ?2AR. 2) To delineate the structural basis for biased conformations of the AT1R by X-ray crystallography and DEER. 3) To determine the structure of GPCR-?arr complexes by cryo-electron microscopy. The insights which we will generate have the potential to guide the design of powerful new cardiovascular drugs and will further our understanding of the conserved signaling mechanisms of the greater GPCR family.